The role of the bla KPC gene in antimicrobial resistance of Klebsiella pneumoniae.

Background and Objectives
Klebsiella pneumoniae isolates that produce K. pneumoniae carbapenemase (KPC) have become a grave concern for the treatment of infections. KPC-producing strains are not only able to hydrolyze carbapenems but are also resistant to a variety of β-lactam and non-β-lactam antibiotics. The present study evaluated the prevalence of bla KPC in K. pneumoniae infections and determined the antimicrobial susceptibility of the isolates.


Materials and Methods
The K. pneumoniae isolates were identified by biochemical tests and confirmed by genotyping. The modified Hodge test (MHT) was performed to detect carbapenemases, and antimicrobial susceptibility was determined for all isolates by the disc diffusion method. Also, for MHT-positive isolates, supposed to carbapenemases isolates, broth microdilution method was used to measure the minimum inhibitory concentrations (MICs) of meropenem and colistin.


Results
The bla KPC genotypic evaluation revealed that only 5 of 96 isolates carried bla KPC genes. Antimicrobial pattern showed that isolates carrying bla KPC were resistant to cefepime, ticarcillin/tazobactam, and aztreonam discs. Also, results of broth microdilution method showed that KPC-producing K. pneumoniae was resistant to meropenem and colistin, according to the CLSI and EUCAST.


Conclusion
In this study nearly half the isolates showed carbapenemase activity as shown by MHT results, but only few of them were carrying bla KPC. Thus bla KPC gene is not the main cause of resistance spread to carbapenems in Isfahan, Iran.


INTRODUCTION
Klebsiella pneumoniae can infect the urinary tract, respiratory tract, surgical sites, and bloodstream, causing severe diseases such as pneumonia, sepsis, and bacteremia (1). K. pneumoniae with multiple drug resistant (MDR) pattern has a high prev-alence, and the global emergence and dissemination of K. pneumoniae strains resistant to carbapenems has increased (2)(3)(4)(5). Carbapenems as another β-lactam structure are recommended as first-line therapy for Enterobacteriaceae infections, such as K. pneumoniae. Also, carbapenems are increasingly used as the first-line antibiotics for unidentified microorganisms and last-line antibiotics for the treatment of infections (1). Resistance to carbapenems is characterized by one or several mechanisms, including carbapenemase production. Carbapenemases exhibit the highest antibiotic resistance, as they can hydrolyze carbapenem antibiotics and other β-lactam antibiotics (6).
Klebsiella pneumoniae carbapenemase (KPC) is one of the main cause of resistance to carbapenems in K. pneumoniae, and it can widely spread due to its location on various plasmids (7)(8)(9)(10). KPC enzymes constitute a major family of class A serine carbapenemases, which are mainly produced by K. pneumoniae. KPC is, in fact, the most common cause of carbapenem resistance in Enterobacteriaceae (7,11,12). KPC producing K. pneumoniae (KPC-Kp) has affected many countries, especially Greece and Italy, and has rapidly become endemic in some countries, such as China, Colombia, and Puerto Rico (13)(14)(15)(16)(17)(18). Dissemination of KPC around the world highlights the role of bla KPC gene in the spread of antimicrobial resistance; Thus, strains harboring bla KPC gene are a major cause of concern for healthcare systems around the world (19). Therefore, in this study, we aimed to evaluate the prevalence and antimicrobial susceptibility of KPC-producing K. pneumoniae among patients in Isfahan, Iran.

MATERIALS AND METHODS
Bacterial collection. K. pneumoniae isolates were collected from Al-Zahra hospital, Isfahan, Iran between February and June 2016. The clinical specimens included urine samples, wound swabs, peritoneal fluid sample, blood samples, respiratory secretions, and others (20).

PCR detection of Klebsiella pneumoniae.
After biochemical test for detection of isolates, polymerase chain reaction (PCR) based on 16S-23S rDNA internal transcribed spacer (ITS) was performed for the identification of K. pneumoniae isolates using specific primers (forward, ATTTGAAGAGGTTGCAAAC-GAT; reverse, TTCACTCTGAAGTTTTCTTGT-GTTC). The procedure was performed as follows. Five colonies from the overnight culture in trypticase soy agar (TSA) medium were suspended in 100 μl distilled water. For DNA extraction, boiling lysis method was used. Cell debris was centrifuged at 13684 RCF for 3 minutes. Supernatants were used as the source of template DNA for amplification. The cycling conditions were as follows; 10 min at 94°C, followed by 35 cycles of 30 s at 94°C, 20 s at 57°C, and 20 s at 72°C, then 10 min hold at 72°C. Ten microliter of the product was detected by 1% (w/v) agarose gel electrophoresis in 1X TAE buffer (20,21).

Phenotypic confirmation of KPC.
According to the CLSI guidelines, MHT was performed for the detection of carbapenemases. Due to the low specificity of MHT method, both meropenem and ertapenem discs were used separately as the substrates for MHT (20,22).

Genotypic confirmation of KPC. PCR was run
for MHT-positive strains to screen the presence of bla KPC gene. K. pneumoniae ATCC BAA 1705 and K. pneumoniae ATCC BAA 1706 were used as the quality control strains. The forward and reverse primers were KPC-F: ATGTCACTGTATCGCCGTCT and KPC-R: GCTGTGCTTGTCATCCTTGT, respectively. The primers were used at a concentration of 1 µM. PCR was performed as follows. Initial denaturation at 95°C for 5 min, followed by 35 cycles of denaturation at 95°C for 60 s, annealing at 53°C for 30 s, elongation at 72°C for 90 s, and a final extension at 72°C for 10 min. The amplification product was approximately 819 bp in length (20).

KPC isolates.
A total of 54 isolates showed positive results, 37 isolates were classified as carbapenem-resistant using MHT with the meropenem disc, and 47 with the ertapenem disc (some isolates showed positive result with the use of both discs) (20). Five out of 54 Carbapenemase-producing isolates harbored bla KPC genes, as detected by PCR (20).
Disc diffusion assay for KPC-Kp isolates showed that 4 out of 5 isolates were resistant to imipenem, ceftazidime, ciprofloxacin, and gentamicin. All KPC-producing isolates (5 isolates) were resistant to cefepime, ticarcillin-tazobactam, and aztreonam. On the other hand, 3 out of 5 KPC-Kp isolates were sensitive to trimethoprim-sulfamethoxazole. One out of 5 KPC-Kp isolates was sensitive to gentamicin, whereas 1 out of 5 KPC-Kp isolates showed intermediate to ciprofloxacin.

DISCUSSION
The prevalence of infection with KPC-producing K. pneumoniae has generally increased worldwide. Sporadic, epidemic, or endemic isolation of KPC-Kp has been reported. While sporadic outbreaks occurred in France, Spain, Germany, Ireland, and Puerto Rico, endemic cases were reported in USA, Colombia, Greece, and Italy (13,(25)(26)(27)(28). In fact, KPC-Kp strains have become the major MDR pathogens, causing nosocomial infections (27). In this study, molecular epidemiology of bla KPC gene in K. pneumoniae indicated a low prevalence in Iran.
One study indicated that two of the most effective antibiotic groups for KPC-Kp are polymyxins,  (28). Previous reports indicated that KPC-producing isolates are commonly resistant to many antibiotics such as colistin, and carbapenems (29)(30)(31). Also, some studies demonstrated that KPC-producing isolates are susceptible to gentamicin and colistin and intermediate or sensitive to tigecycline (29,30,32). However the present findings showed that all KPC-Kp isolates were resistant to colistin and that most of the isolates were resistant to gentamicin. Also, KPC-Kp showed resistance to imipenem, ceftazidime, ciprofloxacin, gentamicin, cefepime, ticarcillin-tazobactam, and aztreonam discs.
In this study, the disc diffusion assay of all isolates showed that the highest antibiotic resistance was related to imipenem, ciprofloxacin, and cefepime, whereas highest antibiotic susceptibility was related to trimethoprim-sulfamethoxazole. Most of the MHT positive isolates were resistant to colistin. Whereas the MIC of meropenem against 28 MHT positive isolates (non-KPC-producing) exceeded 64 mg/l.

CONCLUSION
The present study revealed sporadic outbreaks of KPC-producing K. pneumoniae. It is important to control the spread of this infection by investigation specific mechanisms of carbapenem resistance and employing effective methods such as active surveillance in health centers (for decreasing health associated infections). Also, the results confirmed the importance of implementing infection control programs to identify and prevent the prevalence of oth-